ProjectDescription Thebroadobjectiveofthisproposalistounderstandthemechanismofhomologousrecombinationandits relationshiptochromosomemaintenanceinbothBacteriaandEukarya.Ourapproachistoreconstitute increasinglymorecomplexreactionsthatrecapitulatethestepsofthehomologousrecombinationprocess, usingpurifiedproteinsfromEscherichiacoliandfromSaccharomycescerevisiae,andtousesophisticated biochemicalandbiophysicalmethodsofanalysis.Inparticular,wewillusesingle-moleculevisualizationto imagekeystepsandintermediatesofthesecomplexbiologicalprocesses.Wehavedevelopednew experimentalstrategiesforstudyingprotein-DNAinteractionsatthesingle-moleculelevel.Wedevisedseveral approacheswhereinwecouldeasilymanipulateanindividualDNAmoleculeandexposeittoavarietyof differentproteins,enzymes,andbuffers.Usingfluorescentdetection,wecanimageindividualproteinsonDNA ordetecttheiractiononindividualDNAmolecules,typicallyinreal-time.Weproposetocontinuetousethese approachestostudyunresolvedaspectsofrecombinationalDNArepairanditsinterfacewiththeimportant biologicalprocessesofchromatinremodelingandDNAreplication.Weseektocontinuetoaddressunresolved questionsregardingthedynamicbehaviorofRecA/Rad51nucleoproteinfilamentsandtheirregulationby proteinmediatorsandmodulators;?themechanismbywhichRecAandRad51findDNAsequencehomology;? thereconstitutionincreasinglymorecomplexrecombinationprocessesusingpurifiedproteins;?themechanism andfunctionofDNAhelicasesandtheirpartnerproteins;?andunderstandingthemechanismofDNA transactionsthatintersectwithrecombination,namely,chromatinremodelingandDNAreplication.